1. Field of the Invention
The present invention relates to a single molecule detection technique, and more particularly, to a single molecule detection platform, the manufacturing method thereof and the method using the same.
2. Description of the Related Art
Enzymes are catalysts existing in organisms for performing a variety of crucial tasks and functions. Much scientific research focuses on the structure and functions of enzymes. For centuries, most observations of enzymes have been obtained by taking whole-scale measurements of very large amounts of bio-molecules. However, such measurements fail to provide reaction kinetics of single molecules.
Recently, nanotechnology has become one of the most important fields of research. From high-tech industries to ordinary consumer apparatuses, applications of nanotechnology can be seen almost everywhere. Nanotechnology can be applied to a variety of fields, such as the basic science research or applied science research, including biology, pharmacy, physics, chemistry, material engineering and optical engineering. Nanostructures can also be found in nature, such as the epidermal cells of the lotus leaf that exhibits the lotus effect, or all kinds of insects with nanometer-scaled magnetic particles inside their bodies.
Recently, single-molecule nanotechnology that integrates biology and nanotechnology has been widely utilized and is expected to overcome the issues of performing whole-scale measurements, and accordingly provides an opportunity for starting a new era of biology. Such technology allows researchers to study the reaction behavior of single molecules in real time. However, although current development of single-molecule nanotechnology provides methods to study the characteristics of single molecules, most of those methods are realized by controlling the sample concentration and monitoring the reactions among a vast number of bio-molecules. Accordingly, even if observing large quantities of samples, researchers can only obtain limited information. In addition, additional statistics are required to clarify the ambiguities of the single molecule reaction.
Take, for example, a design experiment for the mutual reaction between an enzyme and a substrate. Such design experiment uses enzyme-containing grooves of 50 nanometers to limit the contact between the enzyme and substrate such that the reaction probability of the enzyme and substrate is reduced. Such design experiment improves the detection concentration of the substrate a thousand times compared to the conventional methods. In addition, the limited volume of the grooves is helpful for the excitation of the local fluorescence of the reaction. However, such design experiment suffers from several drawbacks:
1. The single-molecule reaction between a single enzyme and substrate is not guaranteed. In other words, the probability of the occurrence of the single molecule reaction ranges from one in a hundred to one in ten thousand, and thus total control is difficult to achieve. If the mutual reaction between the enzyme and substrate takes two or three steps, such drawback becomes more significant.
2. To clarify the ambiguities of the single-molecule reaction, countable statistics, which require large amounts of experiment data and time, are required.
3. If the concentration of the substrate is low, it may take too long for the single-molecule reaction to occur. This drawback may greatly reduce the probability of the second and the third steps of the reaction between the enzyme and substrate, and results in the limitation of the capability for measuring the reaction kinetics of the enzyme.
4. To maintain a reasonable reaction probability and detection time, the range of the substrate concentration must be controlled within one or two orders.
Accordingly, there is a need for a system and method that can overcome the aforementioned drawbacks and can increase the range of the molecule reaction kinetics, reduce background noise, increase the controllability of local substrate concentration, and reduce the range of the excitation signal.